1. Field of the Invention
The invention relates to a homogeneous gene probe test, which is based on altering the signal of the non-hybridized gene probe by means of a receptor directed against the label.
2. Description of Background Art
Gene probe assays already have been described in the literature in various embodiments. More frequently used embodiments are the hybridization protection assay (Clin. Chem. 35/8, 1989, 1588-1594), the kissing probes technique (Nachr. Chem. Tech. Lab. 37/7, 1989, 698) and the energy transfer principle.
A very general principle of a gene probe assay according to the prior art is represented in FIGS. 1(a)-1(c): in the first step, target sequence and labeled gene probe are hybridized with one another, wherefrom double-stranded constructs result if there is sufficient homology of the two sequences. Moreover, as a rule, however, non-hybridized single-stranded portions of the gene probe also remain. In the second step, a selective hydrolysis is carried out, which comprises, on account of the conditions selected, essentially the label of the single-stranded gene probe being attacked, while the label of the double-stranded construct is largely protected from hydrolytic attack. Thus in the third step essentially the signal produced by the label bound in the double-stranded construct is then measured.
A disadvantage of the above method is that, despite the treatment with the selection reagent (step 2), a remnant of single-stranded gene probe having an intact label remains, which distorts the measurement.
The present invention is therefore based on the object of making available a method for the determination of a nucleic acid sequence (a "gene probe assay") in which the non-hybridized labeled gene probe contributes to a smaller extent to undesired signal formation than in the method according to the prior art. In particular, the improvement aimed at should make possible an improved homogeneous test procedure. The homogeneous test procedure is fundamentally characterized by the absence of a physical separation step between the nucleic acid hybridization and the signal detection. In such a method, according to the prior art, a particularly severe interfering effect has to be taken into account due to the non-hybridized labeled gene probe.
The object was surprisingly achieved by employing in the method according to the invention a receptor which can bind to the label and as a result of the binding detectably alters, for example attenuates ("quenches") the signal to be attributed to the label. By means of the method according to the invention described below, it is therefore possible significantly to reduce the interfering effect due to the non-hybridized labeled gene probe and thereby to improve the sensitivity and specificity of the test system decisively.